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SCANNING ELECTEON MICROSCOPE STUDY OF THE EESPOHSE OF THE LA~ AND TRA,,C~ 0r RAT TO SMOKE EXPOSUEE REPORT NO, RD. 1552 RESTEXCTED 28.12. 1977 AUTHOR: G. Smlth ISSUED BY: S.E. Evelyn PEOG. EEF. : 11. O 1. IO DISTR~BLr/ION: Dr. S.J. Green Dr. 1.W. Hughes Dr. R.A, Sanford R.M. G£bb, Esq. R.S. Wade, Esq. R.G. Nicholls, Esq. ~rr E. Rittershaus Dr, F. Seehofer Dr. C,J.P. de S£qu/era Dr. D.G. Feluon Library Copy No. 1 M I| 2 " " 3, 4 " "' 6, 7, 8 " " 9, 10 " " 11 " " 12 " " I3 " " 1~ " " 15, 16 COPY NO.: O'- C~ r~ emm~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

BAT Co LTD - MINNESOTA TOBACCO LITIGATION

CSIC.,AL/460 Group Research & Development Centre, British-American Tobacco Co. Led., SO--TON. 28ch December 1977 SCANNZNG ELECTRON MZCEOSCOPE STUDY OF THE RESPONSE OF THE lArYNX AND TRACHEA OF TEE RAT TO SMOKE E~OSORE (Keporc No. 1U).1552 Kescr£cted) SUMMARY AND CONCLUSIONS Groups of rats were exposed for up to 3 weeks to A different concennratlons of smoke. Zn all exposed animals chars was a conslstenc smoke-induced change in the lar~ax, with columnar cells on the inner surface of the vocal cords being replaced by squamous cells. This chanKe was quantifiable and related to length of exposure. For exposure periods of 2 weeks and less there was no clear dose- related response, buC after 3 weeks the race exposed Co the 3 hizhest smoke concentrations produced a sLudlar maximum chanKe. Race exposed to the lowest concentration of smoke invariably showed a decreased response. The nature of che 1salon formation and its deveZopmsnt are d/mcussed. The study indicates thaC a minimum exposure of acclimatisaclon perLod plus 3 to h weeks is probably requ/red for satlsfactory Inhalation bioalsay experiJnmnts based on measurement of 8moke-£nducad lesions of the larynx. BAT Co LTD - MINNESOTA TOBACCO LITIGATION O% r',o o%

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-2- ZNTEODUCTZON The rapid response of the rat larynx Co smoke is thought to be due, at least in part, to the lability of the non-ciliated columnar cells that form a transition area between the equamous and ciliated epithelia of the slottis. In a smoke-exposed rat these columnar cells have been shown to be readily replaced by the £1attened squamous cells which, with continued exposure, become hyperplastlc. There is thus not only a change in the cell type (metaplasia) but also an increase in cell numbers and hence in epithelial thickness (hyparplasla). To study the progression of squamous metaplasia only, using conventional histological techniques, requires the preparation of serial sections which are both tedious and time consuminE to produce. The scanning electron microscope offers a practical, relatively straightforward alternatlve means of obsaEving the majority of the columnar eplthelium present ~n a single larynx. ~nce any smoke-induced changes that may occur in this region can be readily evaluated hy this technique. Squamous hyperplasia, following metaplasia, is not observable usinE the S.E.M. but can be mlasured by routine histolo&ical techniques. The extent of squamoue metaplasia on the inner surface of the vocal cords has been shown to be related Zo both averaBe concentration of the dilute smoke and the length of the exposure period of up to 6 weeks (I). The developwent of the lesion on the. floor of the larynx is rapid and no reproducible accurate method of quantifying the changes has yet been found. The particular a/~ of t~ present investigation was to examine smoke-induced squamous ~taplasia produced on the vocal cords of rats CY~ C~ r~D BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-3- exposed Co 4 concentrations of smoke. The york was extended slightly co include the examination of the ciliated epLthelium o£ the upper trachea. MATERIALS AND METHODS Yemale Wiscar rats (CFHB strain, supplied by Anglla Laboratory Animals) were exposed Co dilute smoke on a 2e-port B.A.T-Mason smoke inhalation machine. Standard flue-cured filter ciKarecces (Code J54) gave a mean delivery of 18.0 mg T1qi/cisareeCe and burned down to a standard butt length of 25 mm in 9 puffs. Animals were exposed twice a day (4 hours between exposures) on week days and once per day on Saturday and Sunday. The normal 7 day acclimatieaeion schedule was followed by exposures of 1 day, 1, 2 and 3 weeks, ac & smoke dilutions providing a ranks of everaKe chamber smoke concentrations (1:8 dilution, 7.0 mg/£; 1:12 dilution, 4.8 mS/E; 1:16 dilution, 3.5 mS/l; and 1:22.5 dilution, 2.6 mg/£). Autopsies were undertaken the day after their final scheduled exposure. The larynx and upper trachea were removed and prepared for the S.E.M. (2). On each P0St-mortem day corresponding sham-smoked controls were killed. There were 10 race per group and a cecal of 20 SToups. There were no pre-scheduled deaths. RESULTS The overall pattern of the larynseal epiChel£e was not examined in dens/1 here but the relative distribution of th~ 3 types present was as described previously for control of rats of similar eke and strain, The two main eplthel£al types, squamous and cilleCed, were separated by a narrow band of non--cilleCed colt-m~sr cells in the Klottis (2). Exposure Co dilute smoke reduced the e~Cmnc of the non-ciliated columnar c~lls mmmm~ O% O BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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--4- due to a 8make-induced squmnous menaplasia. This ~8s observed on the floor o£ the larynx and ou the vocal cords. The ventral depression was not included in the lazTnx preparation, but previous work has shown no observable changes in this region due to smoke exposure. All smoke-- induced chanees were so consistent that a smoke-induced larynx could be readily recognised, especially at the higher concentrations o£ smoke. In order to quantify the rate o£ developmant o£ metaplasia, the width of the band of squamous epithelium on the vocal cord was measured. This value was taken along a line at right angles to the leading edge o£ the vocal cord at a specific site (Fi~are 1). This araa is particularly useful as it can be located accurately and the width o£ the equamoue epitheli~ in this area is falrly constant in the control rats (Figure 2). In the 8ha~-smoked control ~oup8 the width of the eplr~elium increased only slightly over the 3 week period. The width of the 8qu~mous epithelium in all rats exposed to dilute snake was consistantly greater than in corresponding sham-smoked control animals (Yij~tre 2). After 7 days acclimatisation to smoke plus 1 day of "full" e~posuze there was a marked increase in the iridth of equ~nous epithelium on the vocal cord, the change being si~lar for all 4 dilutions of e~oke used, particularly at 1=8, 1:12 and 1:22.5. Concinuad ~wposuxa to smoke at the smoke dilution level of 1=22.5 (2.6 ~/Z) for up to 3 weeks produced no further cons'fat:eriC increase in epithelial width. However, the initial increase produced a~ter I day was mmintained throuahouc the exposure period. Similarly, at a smoke dilution of I:12 (4.8 rag/Z) for an exposure period o£ up to 2 weeks the initial rapid increase was also maintained. At this smoke dilution, continued exposure to smoke BAT Co LTD - MINNESOTA TOBACCO LITIGATION m O O'~

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